Team:UCLA/Notebook/Protein Cages
Contents
Goals
The goal of this project is to produce various mutants of a 3-dimensional protein fusion capable of self-assembling into a tetrahedral cage structure (PDB: 3VDX). These variants will have thrombin protease sites (LVPRGS) introduced in selected locations, allowing for dissociation of the cage structure upon thrombin treatment. Ultimately, we aim to develop a controllable system allowing for both drug-loading and release using the protein cage scaffold.
Achievements
We used Pymol software to model our protein cage and determine where to insert out thrombin cut site mutation.
Using the design restriction listed below, we narrowed down 13 possible locations for the insert
We successfully cloned and expressed one of our mutant cages and our wild type cage.
We ran dynamic light scattering on both but did not see correct size cage formation.
Design
Following is a list of our insertion sites. Sites marked with asterisks are preferred sites. Sites were selected by examining the DSSP secondary structure to ensure minimal disruption of existing alpha-helices or beta sheets, ensuring sites were sandwiched by glycine residues, and using PyMOL to check if the site would be accessible to the protease. Top candidates are based on two main criteria:
1. Geometric accessibility by protease
2. Speculated extent of conformational changes by insertion (minimum preferred)
PyMOL PSE file with sites highlighted: https://drive.google.com/file/d/0B7kb5ShZyyqVcU0xWlVYbUVfSW8/view?usp=sharing
| Site Number | Residues | Original Sequence | Mutant Sequence | Notes |
|---|---|---|---|---|
| Site 1* | 298-305 | IPSGPLKA | IPSGLVPRGSGPLKA | Satisfies 1 and 2, depending on oligomerization geometry |
| Site 2* | 134-140 | DNPDGAA | DNPDGLVPRGSGAA | Satisfies 1 and 2 |
| Site 4* | 252-258 | VEGAPHG | VEGLVPRGSGAPHG | Satisfies 1 and 2; is close to linker region |
| Site 5* | 331-336 | TRPILSP | TRPGLVPRGSGILSP | Satisfies 1 and 2, depending on oligomerization geometry |
| Site 10* | 243-248 | ALPSAE | ALPGLVPRGSGSAE | Satisfies 1, possibly 2 also, very exposed for thrombin cleavage |
| Site 13* | 351-354 | VPSE | GLVPRGSGE | Satisfies 1; only 4 insertions needed. |
| Site 6 | 228-233 | DRTLPI | DRTLGLVPRGSGPI | Satisfies 1 |
| Site 7 | 216-220 | IDVPA | IDVGLVPRGSGPA | Satisfies 1, possibly 2, easily accessible and within longer turn |
| Site 8 | 64-72 | SSQPTTGYD | SSQPTTGLVPRGSGYD | Satisfies 1, but not ideally oriented for access by thrombin |
| Site 9 | 417-422 | RMGAVT | RMGLVPRGSGAVT | Satisfies 1, depending on geometry, possible steric hindrance (inner portion of cage) |
| Site 11 | 123-129 | ASLEPFL | ASLGVPRGSGEPGL | Sterically hindered. 6 insertions needed. |
| Site 12 | 318-321 | KNTD | KNGLVPRGSGTD | Satisfies 1, 7 insertions needed. |
| Site 14 | 190-196 | AASGGFF | AASGLVPRGSGFF | Satisfies neither 1 nor 2, sterically hindered and close to secondary structures. Probably not good. |
Cloning
Have not started yet. Will begin designing gBlocks and primers for site-directed mutagenesis early next week.
gBlocks designed and primers designed to amplify sequence and to add 6x His tag so that sequence can be cloned into vector with T7 and RBS.
PCR was done but we're getting nonspecific binding and are currently troubleshooting and optimizing.
Mutant 10 cage was successfully cloned.
Protein Expression
Long way to go till we get here.It's here!
We are expressing protein from the constructs that Yeates gave us.
We have expressed the wild type cage and the mutant 10 cage.
What we are working on now
We are currently narrowing and refining our list of selected mutation sites.
We are currently troubleshooting and optmimzing our PCR to optimize yield.
We look to get the yield we one and to start cloning into the T7/RBS igem vector.
We are currently protein expressing constructs that Yeates gave us and will be characterizing cage structure soon.
We are cloning our 9 other mutant cages and are planning to express them.
We are looking for cage formation at the appropriate size of our native and mutant 10 cages.
We will introduce thrombin and look for cleavage of our mutant protein cage structures.
Raw lab notebook entries
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